Two of the most promising approaches to cancer immunotherapy are immune checkpoint blockade by monoclonal antibodies and the adoptive transfer of immune cells. The recent successes in treating melanoma with anti-PD1 and anti-CTLA antibodies as well as the extremely successful application of T cells engineered to express the CAR receptor targeting CD19 to treat B cell lymphomas initiated a waterfall of research in identifying new indications and targets for both antibodies and immune cells. While highly effective in treating hematological malignancies, adoptive transfer of natural or engineered T cells is much more challenging to implement for the treatment of solid tumors, in large part due to the immunosuppressive tumor microenvironment. Advirna is proposing to improve the efficacy of adoptive cell transfer therapies by pre- treatment of immune cells ex vivo with RNAi reagents to prevent immunosuppression. Our approach is based on the use of unique, self-deliverable RNAi (sdRNAi) molecules that have been chemically modified, conferring them stability and the inherent ability to enter immune cells, without the need for exogenous reagents or methods. Because of its simplicity, self-deliverable RNAi technology is perfectly compatible with existing cell growth protocols (sdRNAs are used as media component) and is very easy to implement. sdRNAi high efficiency of delivery (>99%), lack of toxicity and strong and long-lasting effect of gene suppression enables knockdown of multiple immune checkpoint and/or regulatory molecules involved in immunosuppression. This may protect the adoptively transferred immune cells in the tumor microenvironment and enhance their efficacy as antitumor agents. sdRNAs can be applied to a broad variety of ACT technologies (CAR-T, engineered TCR, TIL, NK). The unique properties of sdRNAi technology makes it a method of choice for multiplexed immune check point modulation in CAR-T cells. We have generated lead sdRNA compounds targeting a number of immune checkpoints (PD1, LAG3, TIGIT etc.).Our preliminary results in tumor infiltrating lymphocytes (TILs) (enhancing their cytotoxicity to melanoma tumor cells) and in mesothelin targeting CAR-T cells (increasing their efficacy in a mouse model of ovarian cancer) show promise for the success of the approach. We propose to further optimize the existing chemical modification pattern of sdRNAs to improve the knockdown efficiency and longevity of the RNAi effect, and to evaluate the effect of simultaneous knockdown of multiple immune checkpoints in vitro and in vivo using mesothelin CAR-T cells. Our overall goal is to create a series of optimized sdRNAs targeting most of the known immune checkpoints to allow customizable combinatorial RNAi ?cocktails? for specific tumor and ACT cell types to protect immune cells and increase their efficacy. Future in vivo studies in collaboration with academic groups and biotech companies will further evaluate this concept with a goal to move this technology to clinic as a general approach for enhancement of efficacy of any cell-based therapies to treat solid tumors.